Báo cáo khoa học: Post-ischemic brain damage: targeting PARP-1 within the ischemic neurovascular units as a realistic avenue to stroke treatment pptx

Post-ischemic brain damage: targeting PARP-1 within the ischemic neurovascular units as a realistic avenue to stroke treatment Flavio Moroni and Alberto Chiarugi Department of Preclinica

Post-ischemic brain damage: targeting PARP-1 within the ischemic neurovascular units as a realistic avenue to

stroke treatment

Flavio Moroni and Alberto Chiarugi

Department of Preclinical and Clinical Pharmacology, University of Florence, Italy

Therapeutic strategies aimed at reducing brain

dam-age after ischemic stroke have been a major focus of

academic and industrial research for the past

30 years Two primary therapeutic approaches have

been intensively studied: the first can be defined as

the ‘vascular approach’ and its main goal is the rapid

re-opening of occluded blood vessels so that oxygen

and nutrients may return to the ischemic region The

second may be defined as the ‘cellular approach’ and

is based on the possibility of interfering with the

sig-naling pathways, leading to loss of neurons and

dam-age of other cellular elements present in the affected

brain region [1,2] Efforts directed at developing

effec-tive vascular therapy led to clinically useful

procedures and have clearly demonstrated that it is

possible to reduce, selectively, brain damage and

neurologic disability by administering recombinant tis-sue plasminogen activator within 3 h from when the stroke symptoms first start Conversely, the cellular approach has been so far clinically unsuccessful, and none of the numerous neuroprotective strategies that have been tested in clinical trials have reached the clinical arena [3,4]

Exciting, radical, suicidal and inflamed – the many pathways of ischemic brain injury

The enormous body of information on ischemic neuro-degeneration in different experimental stroke models has shed light on the complex signaling pathways and molecular events responsible for neuronal damage

Keywords

blood brain barrier; endothelium;

inflammation; ischemia; microglia;

neuroprotection; neurovascular unit;

PARP-1; pericytes; stroke

Correspondence

F Moroni, Dipartimento di Farmacologia,

Viale Pieraccini 6, 50139 Firenze, Italy

Fax: +39 055 4271226

Tel: +39 055 4271280

E-mail: flavio.moroni@unifi.it

(Received 3 July 2008, revised 11

September 2008, accepted 14 October

2008)

doi:10.1111/j.1742-4658.2008.06768.x

Stroke is the third leading cause of death in industrialized countries but efficacious stroke treatment is still an unmet need Preclinical research indi-cates that different molecules afford protection from ischemic neurodegen-eration, but all clinical trials conducted so far have inexorably failed Critical re-evaluation of experimental data shows that all the components

of the neurovascular unit, such as neurons, glia, endothelia and basal mem-branes, must be protected during the ischemic insult to obtain substantial and long-lasting neuroprotection Here, we propose the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) as a key effector of cell death in the various elements of the neurovascular units, and assert that drugs inhibiting PARP-1 may therefore represent valuable tools for pharmacolog-ical treatment of stroke patients

Abbreviations

AIF, apoptosis-inducing factor; BBB, blood–brain barrier; HMGB1, high-mobility-group protein box 1; IL, interleukin; MMP, matrix

metalloproteinase; NMDA, N-methyl- D -aspartate; PARG, poly(ADP-ribose) glycohydrolase; PARP, poly(ADP-ribose) polymerase; PARP-1, poly(ADP-ribose) polymerase 1; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-a.

when blood flow to a brain region drops below a

criti-cal threshold and when it returns because of vessel

re-opening and tissue reperfusion In the past,

particu-lar attention was directed to derangement of excitatory

amino acid-mediated neurotransmission that became,

for years, the main target for neuroprotection

Hypoxia⁄ ischemia increases the concentrations of

extracellular glutamate [5,6] with excessive stimulation

of ionotropic and metabotropic glutamate receptors,

which initiates a chain of events leading to excitotoxic

neuronal death [7,8] This concept is strongly

sup-ported by the observation that, in a number of in vitro

and in vivo experimental models of ischemia, glutamate

receptor antagonists, acting either on ionotropic

[N-methyl-d-aspartate (NMDA) or Gk

alpha-amino-3-hydroxy-5-methyl-4-isoxazolone propinate] or on group

I metabotropic receptors, are effective neuroprotective

agents [9–13] Unfortunately, however, none of the

glutamate receptor antagonists tested in clinical trials

showed positive results or had an acceptable benefit⁄

side effects ratio

Triggered by the excitotoxic events as well as by

impairment of mitochondrial respiration, a burst of

reactive oxygen species (ROS) and reactive nitrogen

species typically occurs within the ischemic brain

tis-sue Again, inhibition of radical formation as well as

of radical scavengers provides significant

neuroprotec-tion in animal stroke models Agents acting as

free-radical scavengers therefore have been repeatedly

proposed as useful drugs for stroke therapy, but most

were rapidly discarded because of cardiovascular

toxic-ity Recently, however, the spin-trap nitrone NXY-059

from AstraZeneca reached the clinical arena with some

success [14] The putative neuroprotectant is probably

n-t-butyl hydroxylamine and⁄ or its parent spin-trap

2-methyl-2-nitrosopropane, produced by hydrolysis of

NXY-059 Unfortunately, the positive outcome of the

first clinical trial was not confirmed in a second clinical

trial, and NXY-059 development was dropped, leaving

widespread scepticism in the field regarding the

possi-bility of obtaining ischemic neuroprotection in humans

[15]

Apoptotic mechanisms also contribute to ischemic

neuronal demise This suicidal form of

neurodegenera-tion seems to occur mainly in specific types of brain

ischemia, including the global type of brain ischemia

Also, activation of the apoptotic program typically

occurs in a delayed manner in brain regions present in

the surroundings of the ischemic core (the so-called

‘penumbra’, see below) and is thought to be a key

com-ponent of time-dependent brain infarct evolution [1]

Yet, strategies aimed at inhibiting the several apoptotic

effectors have not been exploited at the clinical level

Another event widely recognized to be of key patho-genetic relevance to post-ischemic brain damage is immune activation of resident glial cells and leukocytes infiltrating from blood vessels [16,17] In this regard, several therapeutic approaches aimed at counteracting post-ischemic immune activation and infiltration have been tested in clinical trials Some, such as the anti-leu-kocyte adhesion molecules enlimonab and HU23F2G, proved inefficacious and harmful, respectively Others, such as the interleukin (IL)-1 receptor antagonist, pro-vided inconclusive results Failure might be a result of the fact that both protective as well as detrimental effects of the inflammatory response during ischemic neurodegeneration have been reported [18]

Critical re-evaluation of drug development in stroke

Preclinical studies clearly show that it is feasible to protect the brain from ischemic injury by means of pharmacological or genetic approaches aimed at tar-geting the molecular mechanisms involved in ischemic neurodegeneration Hence, because there are no appar-ent reasons why these strategies should not be effective

in humans, it is reasonable to predict that effective neuroprotective strategies identified at the preclinical level also reach clinical practice Then, the question is why has this not yet happened? An increasing body of literature is accumulating on this subject, and several critical points that have been identified are the past and, unfortunately, present criteria and methodologies used for drug development in the stroke field [3,4,19]

To summarize, it is now clear that animal models should closely reproduce the complex cardiovascular and cerebral pathophysiology of stroke patients, and neuroprotection should be evaluated on a long-lasting and functional basis, rather than on an acute and his-tological basis Also, careful and rigorous selection of patients with salvageable tissue [evidenced using mag-netic resonance imaging as the presence of an area of hypoperfusion larger than that of altered water diffu-sion (the latter is an index of necrosis), the so-called

‘Perfusion⁄ Diffusion (PWI ⁄ DWI) mismatch’] should

be conducted before treating them with an anti-stroke drug candidate [4] Finally, the concepts of ‘pleiotypic drugs’ (i.e drugs with several mechanisms of action) and ‘synergistic combinatorial drug therapy’ emerge as key requisites for efficacious stroke treatment [4] Indeed, one of the possible reasons for the lack of clin-ical efficacy of drugs tested in clinclin-ical trials for brain ischemia is their selective mechanism of action For instance, glutamate antagonists act exclusively (or pre-dominantly) on neurons So, even if neurons are the

first cell type to lose their function when blood supply

is insufficient, the other cell types present in the

ner-vous tissue are of the utmost importance to support

neuronal functioning When capillaries and glia are

damaged, neurons cannot survive in spite of protection

from excitotoxic insults Similarly, selective blocking of

apoptosis or inflammation within the ischemic tissue

cannot provide protection when the other detrimental

events are unrestricted As a whole, efficacious stroke

treatment needs concomitant targeting of the various

pathogenetic events actively contributing to

neurode-generation in cells localized within the ischemic

penumbra

Penumbra and the neurovascular unit

The ischemic brain region may be divided into a zone

in which blood flow is completely absent (‘ischemic

core’) and a peripheral zone in which collateral

ves-sels supply only a fraction of the oxygen and glucose

required for the normal activity of neural cells

(‘ische-mic penumbra’) [2,20] While all cell types in the core

region undergo typical necrotic features and die

form-ing an infarct zone, the ischemic penumbra may

initially retain its morphological integrity, even if its

functions (i.e electrical activity, synthetic processes,

bioenergetic functions, etc.) are temporally lost

How-ever, if sufficient blood flow eventually returns to the

ischemic region within a reasonable time (hours) it is

possible to rescue this area, thus limiting the

neuro-logical damage It is now clear that in order to

obtain full functional recovery, not only neurons, but

all cell types (i.e astrocytes, microglia,

oligodendro-cytes, endothelial cells, muscle cells, pericytes) and

structures (mainly basal membranes) present in the

‘penumbra area’ should be rescued [21,22] Thus,

ischemic neuroprotection can be achieved only if the

classic, oversimplified strategy ‘save the neurons’ is

changed into ‘save neural and stromal cells’ Overall,

neural and stromal cells are grouped into a functional

entity: the so-called ‘neurovascular unit’ Operatively,

the latter is a very complex network of functions

brought about by different cells and aimed at

main-taining the homeostatic milieu necessary for normal

brain activities Protection of the components of the

neurovascular unit seems therefore essential to reduce

brain damage and neurological deficits after a stroke

To achieve this, different strategies have been

pro-posed and evaluated in preclinical settings Yet,

con-comitant targeting of all the components of the

neurovascular units adds substantial complexity to

the feasibility of obtaining ischemic neuroprotection

by pharmacological approaches and, as mentioned

above, general scepticism permeated the field As outlined below, we claim that poly(ADP-ribose) poly-merase 1 (PARP-1) inhibitors are among the most efficacious protectants of the neurovascular unit currently available

PARP-1 activation and cell death in the neurovascular unit

Poly(ADP-ribose) polymerases (PARPs) are NAD-dependent enzymes that are able to catalyse the trans-fer of ADP-ribose units from NAD to substrate proteins, thereby contributing to the control of geno-mic integrity, cell cycle and gene expression [23] Among PARPs, nuclear PARP-1 is a DNA damage-activated enzyme of 113 kDa molecular mass and is the most abundant and commonly studied member of the family Its enzymatic activity leads to poly(ADP ribose) formation, and it was first described over

40 years ago in liver cell nuclei incubated with NAD and ATP in Paul Mandel’s laboratory in Strasburg [24] Although this seminal observation was made in a neuroscience laboratory, for the following 30 years, research on PARP-1 was exclusively carried out by researchers mainly involved in genome stability, DNA repair and cancer The neuroscience community ignored PARP-1 until the early 1990s when it was shown that it mediates glutamate-induced and nitric oxide-induced neuronal death [25,26] Excellent work carried out in the following years uncovered several molecular events causally linking PARP-1 activation to ischemic cell death [27] As for the triggers of PARP-1 hyperactivity during ischemia, ROS-dependent DNA damage is thought to play a major role However,

Ca2+-dependent and kinase-dependent PARP-1 activa-tion might also contribute [28–30] Ambiguity also exists regarding the molecular mechanisms underlying the detrimental role of the enzyme in ischemic brain injury [31,32] Indeed, although we know in part the mechanisms activated by PARP-1 and triggering neurotoxicity, which of these is causally involved in PARP-1-dependent ischemic neurodegeneration still needs to be elucidated

Experimental data demonstrate that, upon different stresses, activation of PARP-1 can exert detrimental effects in every cell type of the neurovascular unit (Fig 1) Given that the ischemic challenge mimics these stresses, we reason that during brain ischemia PARP-1-dependent cytotoxicity occurs in all the com-ponents of the neurovascular unit It is obvious that triggers, time courses and final effects of PARP-1 activation in endothelial, muscle and glial cells, as well

as in infiltrating leukocytes, are different from those

occurring in neurons Regardless, the hyperactivation

of PARP-1 in each single component of the

neurovas-cular unit triggers dysfunction⁄ cytotoxicity and,

indi-rectly, severely affects the functioning of neighbouring

neurons As a whole, PARP-1-dependent derangement

of the integrity of the neurovascular unit is caused by

the enzyme’s ability to prompt an increase of blood–

brain barrier (BBB) permeability, the release of

pro-inflammatory mediators, mitochondrial dysfunction

and bioenergetic failure, as well as the activation of

specific apoptotic pathways

PARP-1, endothelia and post-ischemic

BBB breakdown

Ischemia causes rapid structural changes and

break-down of the BBB, allowing plasma exudation and

immune cell infiltration, which contribute to ischemic

brain damage [22] Very early after the onset of brain

ischemia, and especially after a reperfusion period,

abundant free radicals are generated in macrophages,

endothelial cells, perycites, astrocytes, microglia and

neurons, causing significant damage to brain capillaries and disruption of the BBB [33] Free radicals formed both inside and outside the vessels prompt genotoxic stress and activate PARP-1 in endothelial cells Under conditions of chronic hypoxia, PARP-1 activation within endothelia triggers cell proliferation and slowly developing brain damage The molecular mechanisms

of cell proliferation include the generation and release

of ROS from NADPH oxidase and mitochondria, sus-tained increase of the cytosolic Ca2+ concentration and finally nuclear translocation of mitogen-activated protein kinase kinase⁄ extracellular regulated protein kinase with cell cycle activation [34] Conversely, during ischemia, PARP-1 hyperactivation causes endo-thelial cell death The latter occurs because of cellular accumulation of the PARP-1 product poly(ADP-ribose), which causes translocation of apoptosis-induc-ing factor (AIF) from mitochondria to the nucleus and activation of a caspase-independent programmed cell-death pathway [35–37] Accordingly, the potent PARP-1 inhibitor, PJ34, administered to rats with transient focal brain ischemia, preserves the integrity

M P M P

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H M G 1

y r o t a m a l f n I s r o t a i d m

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n m u Basal lamina

Fig 1 The role of PARP-1 within the ischemic neurovascular units PARP-1 exerts its detrimental role within the ischemic neurovascular unit by promoting necrosis and AIF-dependent apoptosis in neurons, astrocytes and endothelial cells PARP-1 also plays a key role in immune activation and migration of microglial cells upon different noxious stimuli to the central nervous system The expression of adhesion molecules by endothelial cells is also promoted by PARP-1-dependent transcriptional activation, thereby promoting leukocyte recruitment within the ischemic brain tissue and their detrimental effects on ischemic injury Hence, the pharmacological inhibition of the enzyme exerts ischemic neuroprotection by targeting several pleiotypic events of pathogenetic relevance to post-ischemic brain damage X, adhesion mole-cules ADP-ribose monomers are depicted as black circles binding to the transient receptor potential melastatin-2 receptor.

of endothelial tight junctions and decreases the

expres-sion of the adheexpres-sion molecule intercellular adheexpres-sion

molecule-1, thus limiting leukocyte infiltration and the

subsequent inflammatory damage to the ischemic brain

[35,38] It has also been proposed that post-ischemic

PARP-1 activation contributes to increased expression

of matrix metalloproteinases (MMPs), a group of

zinc-containing proteases with key roles in matrix

degrada-tion and disrupdegrada-tion of capillary permeability during

stoke [39] Indeed, pharmacological PARP-1 inhibition

reduces MMP-9 expression levels in plasma and brain

[40], prevents brain matrix degradation, reduces

delayed increase of BBB permeability and edema

for-mation, preserves endothelial tight junction proteins

and decreases delayed infiltration of leukocytes into

the brain of rats with middle cerebral artery occlusion

[41] The key role of PARP-1 hyperactivation in

endo-thelial dysfunction in experimental models of diabetes

underscores the pathogenetic relevance of the enzyme

to disorders of this key component of the

neurovascu-lar unit [42] Accordingly, gene array studies have

demonstrated that upregulation of inflammatory genes

is hampered in PARP-1) ⁄ )endothelial cells exposed to

tumor necrosis factor-alfa (TNF-a) [43] Taken

together, these findings point to basal PARP-1 activity

as central to homeostatic regulation of endothelial

function, whereas its hyperactivation appears causal

to BBB damage and immune cell infiltration during

ischemia

PARP-1, glia and post-ischemic

inflammatory events

Activation of resident immune cells as well as

infiltra-tion of leukocytes within the ischemic area lead to

excessive release of inflammatory mediators and

ensu-ing worsenensu-ing of brain damage In keepensu-ing with this,

astrocytes, microglia and blood-derived leukocytes

contribute to ischemic neurodegeneration, whereas

immunosuppressant strategies able to reduce the

inflammatory response decrease infarct volumes in

dif-ferent stroke models [16,17] Microglial cells are

resi-dent brain macrophages displaying a ‘resting’ highly

ramified phenotype Upon ischemic challenge, before

neuronal damage can be morphologically detected [44],

microglia assume amoeboid morphology and acquire

phagocytic activity, producing ROS and other

inflam-matory⁄ cytotoxic factors such as nitric oxide,

prosta-noids, TNF-a, IL-1b and MMPs Astrocytes and

infiltrating leukocytes within the ischemic brain tissue

also contribute to the synthesis and release of

pro-inflammatory mediators [17] It is now widely accepted

that the latter are responsible for disruption of the

capillary basal lamina, opening of the BBB and infil-tration of blood-borne leukocytes This prompts a vicious circle comprising waves of release of cytotoxic inflammatory products, cell death and recruit-ment⁄ activation of blood or bystander immune cells Eventually, the neuroimmune response causes collapse

of the structures and functions of the neurovascular unit [16,17,45]

Again, PARP-1 plays a key role in this scenario Indeed, numerous reports demonstrate that PARP-1 activity promotes the neuroimmune response thanks

to its ability to assist transcriptional activation and epigenetic remodeling in immune cells In this light, it has been speculated that ischemic neuroprotection afforded by PARP inhibitors is at least partially med-iated by their anti-inflammatory properties [46] Indeed, PARP inhibitors decrease expression of inflammatory markers⁄ mediators such as CD11b, cyclooxygenase-2, inducible nitric oxide synthase, TNF-a, IL-1b, IL-6, intracellular adhesion molecule-1, interferon-gamma and E-selectin in different models

of neurodegeneration [40,47–55] Remarkably, these molecules actively contribute to ischemic neurodegen-eration A key role for PARP-1 in microglia activa-tion and migraactiva-tion towards injured neurons has also been reported [56] Reduced expression of pro-inflam-matory mediators is probably a result of the fact that inflammatory transcription factors such as nuclear factor-kappaB, activator protein-1 and nuclear factor

of activated T-cells are positively regulated by PARP-1 PARP-1 protein per se, as well as its enzy-matic activity, promote transcription factor binding

to DNA as well as supramolecular complex formation containing several transcription-regulating proteins and RNA polymerase II [23,53,57] These findings taken together may explain why post-treatment with PARP-1 inhibitors reduces the neuroimmune response

in different stroke models [58–60]

Recently, the tetracycline, minocycline, has been proposed as a clinically relevant tool to limit post-ischemic brain damage because of its ability to inhibit microglia activation Minocycline is indeed able to reduce brain infarct volumes in preclinical models [61], as well as neurological impairment in stroke patients [62] Interestingly, it has recently been reported that minocycline is a powerful inhibitor of PARP-1 [63] Whether PARP-1 inhibition underpins the drug’s neuroprotective effects in stroke patients is currently unknown Yet, given that minocycline has been largely used without significant side effects, these observations indicate that acute inhibition of PARP-1

in vivo might be a rather safe procedure and could be proposed to preserve the integrity of the ischemic

neurovascular unit and limit post-ischemic brain

damage in humans

PARP-1 and post-ischemic death in

neurons

Excitotoxicity and PARP-1 activation have been

caus-ally linked since 1994 when it was reported that

gluta-mate increases poly(ADP-ribose) synthesis and causes

a type of cell death that is prevented by both NMDA

antagonists and PARP-1 inhibitors [25,26] The

pro-posed molecular events underlying these observations

include: overactivation of NMDA glutamate receptors

with consequent intracellular Ca2+ influx; and

subse-quent ROS production mainly caused by neuronal

nitric oxide synthase activity, which, in turn, triggers

DNA damage-dependent hyperactivation of PARP-1,

depletion of intracellular NAD and ATP stores, and

neuronal death [26] PARP-1 activation may also occur

in neurons without NMDA receptor activation, as

increases of intracellular [Ca2+] triggered by K+

-induced depolarization or inositol

3-phosphate-recep-tor activation are sufficient to trigger poly(ADP-ribose)

formation [28,64] In keeping with this toxic cascade of

events, neurons obtained from PARP-1-deficient mice

are resistant to NMDA toxicity and to oxygen and

glucose deprivation [65] It was also shown that

NMDA-induced overload of cytosolic Ca2+ not only

activates neuronal nitric oxide synthase in the cytosol,

but is also responsible for mitochondrial ROS

produc-tion [66], which contributes to DNA damage and

fur-ther activation of PARP-1 [67,68] Substantial DNA

damage, evaluated by means of the comet assay, is

present in cells isolated from the rat ischemic cortex or

caudate NMDA receptor antagonists reduce the

extent of the damage and provide ischemic

neuropro-tection, while PARP inhibitors decrease infarct

vol-umes without affecting the severity of DNA damage

[69] These observations suggest that NMDA receptor

channel openings, ROS formation, DNA damage and

PARP activation are sequential crucial steps in the

process leading to neuronal death They also indicate

that stroke protection can be achieved without

reduc-ing DNA damage Energy failure followreduc-ing PARP-1

activation is not only caused by NAD resynthesis but

also by glycolysis block because of NAD depletion,

which results in reduced synthesis of both

glycolysis-derived ATP and mitochondrial energetic substrates

[70] Accordingly, tricarboxylic acid cycle substrates or

extracellular NAD supplementation protect neurons

from excessive PARP-1 activation [71], whereas

PARP-1 inhibitors prevent ischemia-induced NAD+

depletion and reduce ischemic brain injury [72] In

apparent contrast to the hypothesis that PARP-1 worsens ischemic neurodegeneration by reducing ATP levels within the injured tissue, however, ischemia-induced energy derangement is similar in the affected brain areas of PARP-1+⁄ + and PARP-1) ⁄ ) mice, despite the latter showing significant reduction of ischemic volumes [73]

Controversy still exists on the molecular mecha-nisms involved in PARP-1-dependent neuronal death during ischemia In this regard it has been very recently reported that exposure of cultured neurons

to poly (ADP-ribose) is sufficient to trigger nuclear translocation of mitochondrial AIF and cell demise [74] The poly(ADP-ribose)-degrading enzyme, poly (ADP-ribose) glycohydrolase (PARG), should be, in principle, a neuroprotective agent [75] Consistently, PARG-110 kDa) ⁄ ) or PARG+⁄) mice show increased sensitivity to brain ischemia [36,76] Also, PARP-1 activity seems to be essential for AIF release within neurons of the infarct area, and AIF-deficient (Harle-quin) mice are less sensitive to post-ischemic brain damage [77] Data therefore point to PARP-1 activ-ity-dependent AIF release from mitochondria as a key molecular event underlying ischemic neuronal death Interestingly, the ADP-ribose monomers origi-nating from the polymer degradation through PARG might also contribute to neuronal demise by activat-ing transient receptor potential melastatin-2 receptors and massive Ca2+ influx [78,79] Finally, the finding that, when released in the extracellular space, high-mobility-group protein box 1 (HMGB1) promotes the neuroinflammatory response and worsens brain ische-mia [80–82], along with evidence that PARP-1 pro-motes HMGB1 release [83] (but also see [82]), indicate that HMGB1 may mediate, in part, the toxic effect of PARP-1 hyperactivation within the ischemic brain tissue Overall, a wealth of evidence points to the synthesis of poly (ADP-ribose) within ischemic neurons as a crucial event contributing to derange-ment of the neurovascular unit

Conclusion

To reduce brain damage after stroke it is not sufficient

to protect neurons from excitotoxic insults, but it is mandatory to rescue all cellular and structural compo-nents of the neurovascular unit As outlined above, PARP-1 activation during brain ischemia plays a detri-mental role in all cell types of the neurovascular unit Inhibitors of PARP-1 might therefore represent a class

of ‘pleiotypic drugs’, which are considered the most promising tools for pharmacological treatment of stroke Also, the different temporal kinetics of PARP-1

activation within the components of the neurovascular

unit would warrant a significant ‘window of

opportu-nity’ to be harnessed for the treatment of stroke

patients Remarkably, the clinical relevance of PARP-1

inhibitors in stroke treatment is emphasized by the fact

that these drugs are well tolerated by patients enrolled

in clinical trials for treatment of tumor malignancies

or coronary bypass, and that, theoretically, anti-stroke

treatment with PARP-1 inhibitors would require an

acute, 4–6-day treatment This, of course, would

reduce the risk of side effects The latter might be

fur-ther reduced by the forthcoming development of

PARP isoform-specific inhibitors [84] In conclusion,

preclinical and clinical data indicate that PARP-1 is a

very promising target for ischemic neuroprotection,

and PARP-1 inhibitors represent a realistic new avenue

to stroke treatment

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